Changes in total cyanide content of tissues from cassava plants infested by mites (Mononychellus tanajoa) and mealybugs (Phenacoccus manihoti)

Agriculture, Ecosystems and Environment, 12 (1984/85) 35--46 "

35

Elsevier Science Publishers B.V., Amsterdam --Printed in The Netherlands

CHANGES IN T O T A L CYANIDE C O N T E N T OF TISSUES FROM C A S S A V A P L A N T S I N F E S T E D BY M I T E S ( M O N O N Y C H E L L U S T A N A J O A ) A N D M E A L Y B U G S (PHENA COCCUS M A N I H O T I )

D.K.G. AYANRU and V.C. SHARMA 1

Department of Microbiology, University of Benin, PMB 1154, Benin City (Nigeria) Department of Physics, University of Benin, PMB 1154, Benin City (Nigeria) (Accepted for publication 5 July 1984)

ABSTRACT Ayanru, D.K.G. and Sharma, V.C., 1984. Changes in total cyanide content of tissues from cassava plants infested by mites (Mononychellus tanajoa) and mealybugs (Phenacoccus manihoti). Agric. Ecosystems Environ., 12 : 35--46. Dietary and industrial uses of cassava (Manihot esculenta Crantz) are threatened by the known presence of cyanide in the crop and its products. The toxic HCN content of cassava may be altered by some intrinsic and extrinsic factors in and around the plant, but the extent of any shifts in this regard due to stress conditions created by cassava green spider mites (CGM) Mononychellus tanajoa and mealybugs (CM) (Phenacoccus manihoti, now spreading epiphytotically in almost all cassava-growing areas in Africa, is unknown. In this study, we report variations in the total HCN content of leaf, stem and root tissues of six cassava clones differentially infested by these pests.

INTRODUCTION Cassava is o n e o f t h e m o s t e c o n o m i c c y a n o p h o r i c plants. A b o u t 60% o f the caloric i n t a k e by over 30 million Nigerians a n d 3 0 0 million p e o p l e in the tropics and s u b t r o p i c s c o m e s f r o m cassava p r o d u c t s . H o w e v e r , cassava's g r o w i n g e c o n o m i c i m p o r t a n c e as a d i e t a r y staple and an animal feed (Leuschher, 1 9 8 0 ) , as well as its industrial uses ( R o b i n s o n a n d K u t i a n a w a , 1 9 7 9 ) c o u l d be r e t a r d e d b y t h e presence o f c y a n o g e n s in cassava a n d its p r o d u c t s . C y a n o g e n e s i s is t h e f o r m a t i o n o f t o x i c h y d r o c y a n i c acid f r o m c y a n o h y d r i n s in d a m a g e d tissues, and is s t r o n g l y e x h i b i t e d by cassava. T h e phen o m e n o n is o f b r o a d p h y l o g e n e t i c d i s t r i b u t i o n , as it o c c u r s in m o r e t h a n 1 0 0 0 p l a n t species spread over 80 families a n d 3 0 0 genera ( K i n g s b u r y , 1 9 6 4 ; Butler, 1965). Sadik et al. ( u n d a t e d ) screened 88 510 cassava plants for c y a n i d e c o n t e n t a n d r e p o r t e d t h a t m o r e t h a n 99% o f t h e m were highly c y a n o g e n i c . In fact, a c y a n o g e n i c cassava plants have n o t been f o u n d , alt h o u g h breeding and selection e f f o r t s are c o n t i n u i n g (I.I.T.A., 1980). Despite c o n s i d e r a b l e r e d u c t i o n s in the c y a n i d e c o n t e n t o f raw cassava during

36 fermentation and processing, as well as the availability of methods for eliminating HCN from some cassava products, toxic levels of cyanide (> 100 mg/kg dry wt.) in various cassava products are still being reported (Nartey, 1981). Acute cyanide toxicity in animals and humans may cause instant death, while chronic toxicity resulting from prolonged consumption of cassava meals may lead to several serious maladies (Osuntokun et al., 1969). The synthesis and chemistry of cyanide in cassava are well known. In undamaged tissues, cyanide occurs as stable aliphatic glucosides, linamarin and lataustralin, from which HCN is released following enzymatic and nonenzymatic hydrolysis (Nartey, 1968; Butler et al., 1973). However, the biogenesis and accumulation of these and other cyanohydrins may be influenced by extrinsic factors such as weather and soil, and by intrinsic ones such as the concentrations and activities of some metabolites and enzymes in the plant (Nartey, 1981). Therefore, it is reasonable to expect t h a t certain stress conditions inducible by pests may influence the levels of cyanide in cassava. Infestations of cassava by the cassava green spider mites (CGM) and mealybugs (CM) identified and confirmed as Mononychellus tanajoa (Bondar) (Acarina: Tetranychidae) and Phenacoccus manihoti Mat-Ferr. (Hemiptera : Pseudococcidae), respectively, have reached epiphytotic proportions in almost all cassava-growing regions of tropical Africa (Lyon, 1974; Akinlosotu and Leuschner, 1981). CGM cause ~ 60% chlorophyll depletion and about 50% leaf area reductions (Ayanru and Sharma, 1983), while CM induce severe defoliation and serious growth abnormalities (Nwanze et al., 1979). The combined effects of both pests may lead to heavy yield reductions, drastic changes in tuber quality, or even total crop failure. It is hitherto u n k n o w n as to what extent cyanide content of cassava is altered by CGM and CM. In this study, therefore, we report on changes in total cyanide c o n t e n t of tissues of six field-grown cassava clones infested to varying degrees by CGM and CM. MATERIALS AND METHODS

Field experiment Mature stem cuttings of six clones of cassava (Manihot esculenta Crantz), with accession numbers TMS 4488, 30572, TMS/SA 1001, TMS/U 30395, 41044 and 42046, were field-propagated at Ugbowo, Benin City, on 5 June 1982. All except TMS/SA 1001 (a sweet variety) were of the bitter variety. The cuttings were planted 1 m apart in 2-m wide rows to give a plant population of about 6428 ha -1. The experimental set-up was a randomized block design in which two adjacent rows (split plots) containing six stands of a clone per row were replicated four times in each of three blocks. The plots contained a coarse-textured sandy loam soil. Soil samples were

15.02 14.83

60.91 60.15

Coarse 3.52 3.81

Silt (%)

18.50 19.21

Clay (%)

2.13 2.17

Organic matter (%) i

5 flame (Na and K) and atomic absorption (Ca and Mg) photometry.

Extraction methods: l dichromate wet oxidation; 2 equilibrating with N a m m o n i u m acetate; 3m i c r o - K j e l d a h l ; 4 Bray;

July samples November samples

Fine

Sand (%)

C h e m i c a l a n d t e x t u r a l c h a r a c t e r i s t i c s o f soil s a m p l e s f r o m e x p e r i m e n t a l p l o t s

TABLE I

5.00 5.31

pH

80 88

Base saturation (%f

0.68 1.06

Total N (%)3

12.10 18.80

Available P (ppm) 4

0.15 0.20

Na

0.17 0.20

K

2.28 2.69

Ca

0.48 0.47

Mg

Exchangeable cations (meq/100 g)S

CO

38 taken from the plots on 20 July and 23 November 1982. The textural and chemical characteristics of the soil samples (Table I) were determined by the Rubber Research Institute of Nigeria (R.R.I.N.), Iyanomo, Bendel State. Fertilizer was applied to the plots (side-dressing) on 22 August 1982 at the rate of 48/48/68 kg ha -1 of N/P~Os/K20, respectively. From 23 August 1982 to 30 February 1983, a Wambo sprayer (Wambo Gartenbau, Hamburg) was used bi-weekly to apply a 100% soluble concentrate (formulated 20 ml/5 1 of water) of Nuvacron (Monocrotophos) (Ciba-Geigy, Basle, Switzerland) to run-off point to half of the plants in the split plots. Stands of TMS/SA 1001 were neither sprayed nor fertilized.

Pest infestation scores On 3 November 1982 and 11 April 1983, individual plants were evaluated for CGM infestations on a scale of 1 (no apparent infestation) to 5 (severe infestation), with intermediate scales, according to the scoring system of the I.I.T.A. {1979). Also, based on known symptoms of CM, plants were rated for vigour on a modified scale of 1 (no symptoms) to 5 (severe symptoms), according to Atu and Okeke (1981). Stands of TMS/SA 1001 were rated for pest injuries only in April 1983.

Harvesting Tissues for cyanide assays -- leaves, stem, tuber peel (periderm or cortex) and tuber pulp (parenchyma) -- were harvested around noon of each day of harvest, beginning on 12 April 1983. For TMS/U 41044 only, tissues harvested from sets of 20 plants were bulked, washed in distilled water, sliced, and dried in an oven initially for 1 h at 100°C, then for 48 h at 65°C. The dried tissues were powdered in a blender, and stored in sealed containers until analysed. For all other clones that were similarly harvested, cyanide c o n t e n t was determined from samples of bulked fresh tissues within an hour of harvest.

Preparation of linamarase and assay for its activity The test enzyme, linamarase, was prepared from fresh tuber peels of 12-month-old TMS/U 42046 plants, according to the m e t h o d of Ikediobi and Okeke (1980). Three 100-g samples were homogenised with a blender for 10 min each in 300 ml pre-chilled (4°C) 0.1 M acetate buffer of pH 5.5. The homogenates were filtered through kieselguhr, the filtrate was stored overnight at 4°C and then extracted with 2.30 volume of cold acetone over a 2-h period. The resultant extract was centrifuged for 5 min at 500 g. All traces of linamarin and other cyanohydrins (cyanogens) were removed from the supernatant by dialyzing it for 3 days at 15°C against a 0.1 M acetate buffer of pH 5.5 The buffer solution was changed several times during the dialysis period.

39 The dialyzed enzyme extract was assayed for activity, using as substrate a solution of p-nitrophenyl-~-D-glucoside (PNP-glucoside) containing 6.2 mg m1-1 of 0.1 M acetate buffer of pH 6.8. This was done by incubating 1 ml of the substrate with 0.5 ml of linamarase preparation for 1 h at room temperature (25°C). The reaction was stopped after 1 h by adding 2 ml of 0.2 M borate buffer of pH 9.80, followed by 0.5 ml distilled water. Absorbance of the solution was read at 400 nm, using a Corning 253 colorimeter (Bausch and Lomb, Halstead, Essex). A unit of activity of the enzyme was defined as the quantity that caused a change of 10 -4 absorbance per min under the test conditions described.

Tissue extract preparation Cyanogens in dried tissues were extracted by homogenising 20-g samples with a blender for 3 min in 150 ml of 0.1 M sodium phosphate buffer of pH 6.8, while those in 20 g of fresh samples were extracted in 50 ml of 0.1 M HC1. The homogenates were vacuum-filtered through glass-fibre filter paper and the pH of the filtrate adjusted to 6.8 with a 4 N NaOH solution. The extracts were then centrifuged for 3 min each at 500 g, dispensed in 30-ml screw-cap vials and stored at 0°C in a refrigerator until assayed for total HCN contents within 1--2 weeks of extraction.

Determination o f cyanide in tissue extract For the quantitative determination of total HCN (Ikediobi and Okeke, 1980) in an extract and hence in a tissue, 0.2 ml of an extract was incubated for 15 min at room temperature with 1.0 ml of the purified linamarase, followed by the addition of 0.8 ml of 0.2 M sodium phosphate buffer of pH 6.8 in a 30-ml screw-cap vial. Then 5 ml of alkaline picrate solution, containing 5 g of anhydrous picric acid and 25 g of anhydrous sodium carbonate per liter, were added. The solution, sealed up in the vial at all stages of the determination, was further incubated for 5 min in a waterbath at 95°C. After the solution was cooled to room temperature, its absorbance was read in the colorimeter at 490 nm, using a similarly prepared blank solution in which 0.2 ml distilled water replaced the tissue extract. A standard curve of KCN cm -3 vs. absorbance (A490) was drawn, from which cyanide concentrations (mg kg -1) in the tissue extracts were extrapolated. The determinations were repeated at least three times for each sample. Cyanide data and pest infestation scores were analysed statistically, using the comparison of two paired samples method of Snedecor and Cochran (1967). RESULTS A few stands of TMS 4488, TMS/U 41044 and 42046 developed symptoms of cassava mosaic disease (CMD), and were removed from the field

40 plots by roguing. All stands of TMS 30572 and TMS/U 30395 were CMD symptom-free, but every plant in plots of TMS/SA 1001 showed severe symptoms of CMD, in addition to those of CGM and CM. Sprayed and unsprayed stands of all clones had mild and uniform symptoms of brown leaf spot caused by Cercospora henningsii on older leaves. The data on pest infestation scores (Table II) show that symptoms of CGM and CM on the test plants varied considerably. CGM symptoms were conspicuous by September 1982, and were rated 64% higher (P < 0.01) on all unsprayed than on sprayed plants in November 1982. CM symptoms were not detected until November 1982. Hence, during this month, scores of the incidence of this pest from sprayed and unsprayed stands were not different. By April 1983, however, all the clones were heavily infested by both pests. Infestation scores made at this time on all unsprayed plants gave mean values that significantly exceeded (P < 0.01) corresponding scores on sprayed plants by 32 and 37% for CGM and CM, respectively. Stands of all clones (except TMS/U 30395) were heavily defoliated by the pests. This made leaf and succulent stem-samples unavailable for cyanide assay for some clones. In such instances, and in others where bulk samples were not available for cyanide determination, expected figures for cyanide were treated as missing data. In order to ensure that sufficient amounts of enzyme and cyanogens in tissue extracts were used in cyanide assays, it was first necessary to determine the activity of the test enzyme. This was achieved by measuring the absorbance (A400) of the resultant solution obtained after incubating 1.0 ml of a solution containing 6.2 mg of PNP-glucoside with 0.5 ml of the enzyme for 1 h. From an absorbance value of 0.23 so obtained, the activity of the test enzyme was calculated to be 38 units. When 1.0 ml of the enzyme was incubated for 10 min with 0.1 and 0.5 ml of a cassava tissue extract, mean absorbance values (A490) of 0.19 and 0.75, respectively, were obtained. These values remained unchanged when 2.0 ml of the enzyme were incubated with the same a m o u n t of substrate as above. This showed that 1.0 ml of the enzyme contained enough activity to hydrolyse completely all the cyanogens in 0.5 ml of the tissue extract. Therefore, all cyanide assays of cassava extracts were carried out by incubating 0.2 ml of tissue extracts with 1.0 ml of the enzyme for 15 min. This ensured a cyanide detection precision limit of 0.16 pg cm -3 (Ikediobi and Okeke, 1980). For all the clones, sprayed and unsprayed plants did not differ in several aspects with regard to their cyanide content. Over-all mean cyanide contents of entire plants from sprayed stands of TMS 4488 and TMS/U 42046 were similar to those from unsprayed plants. In both plant groups also, leaves generally contained the highest amounts o f cyanide, followed by tuber tissues, while stem tissues were relatively low in cyanide. No relationship was f o u n d between cyanide contents of leaf and tuber tissues of a clone, since high or low leaf cyanide contents were not necessarily accompanied by high or low tuber cyanide contents, and vice versa (Table III).

II

1.30 1.10 __4 1.50 1.10 1.35

3 . 2 5 **3 3.10"* . 4.80** 2.65** 3.92**

60 65 . 69 58 66 . 1.00 1.00 1.00

1.00 1.00 . 1.00 1.32 1.10

1.21 1.12

Unsprayed

0 24 9

17 11

% of sprayed

3.56 2.20 3.55

2.50 2.48

Sprayed

Sprayed

Sprayed

% of sprayed 2

CGM

CM

CGM

Unsprayed

April 1983

November 1982

3.32* 4.12"* 3.11 4.90* 2.93* 4.81"

Unsprayed

27 36 26

31 40

% of sprayed

3.20 3.62 -1.77 2.91 1.85

Sprayed

CM

4.90** 4.83* 4.55 2.81" 4.99** 3.10"*

Unsprayed

39 42 40

35 25

% of sprayed

1 S c o r e s w e r e b a s e d o n scales o f 1 ( n o i n f e s t a t i o n ) t o 5 ( s e v e r e i n f e s t a t i o n ) f o r CGM a n d CM, a c c o r d i n g to I . I . T . A . ( 1 9 7 9 ) a n d A t u a n d O k e k e ( 1 9 8 1 ) . respectively. (unsprayed -- sprayed) X 100 2 % of s p r a y e d = unsprayed 3 M e a n scores f r o m 24 plants; * and ** represent significant differences (P ~ 0.05 and 0.01, respectively) between m e a n s of paired tissues of a clone. 4 Represents no score (missing data).

TMS 4 4 8 8 TMS 3 0 5 7 2 TMS/SA 1001 TMS/U 30395 TMS/U 41044 TMS/U 42046

Test clones

Cassava green spider mites ( C G M ) and mealybugs ( C M ) infestation scores on N u v a c r o n - s p m y e d a n d unsprayed plants ]

TABLE

1793-+197 --2 -3150-+690 420_+99 2226-+630

1 0 0 5 + 1 3 8 *.1 -3150+611 1261+79"* -1978+256

256+0 867-+120 -276+39 125+26 591-+39

Sprayed

Sprayed

Unsprayed

Stem

Leaf

414+60"* 1281+20"* 788-+138 256-+20 361+7"* 256_+0*

Unsprayed 453+60 1064+99 -1300-+158 -1970+390

Sprayed

T u b e r peel

553+39 1162+60 2660-+1537 --709-+ 1 5 8 " *

Unsprayed

355-+39 650-+79 -768+99 533-+13 1123-+99

Sprayed

Tuber pulp

433-+79 827-+99* 1340-+79 1793-+20"* 722-+33* 2364-+394**

Unsprayed

* Means w i t h s t a n d a r d deviations f r o m t h r e e r e p l i c a t i o n s ; * and ** r e p r e s e n t significance at 5 and 1% p r o b a b i l i t y levels, r e s p e c t i v e l y , b e t w e e n m e a n s o f paired tissues f r o m s p r a y e d a n d u n s p r a y e d s t a n d s o f a clone. 2 R e p r e s e n t s an i n s t a n c e w h e r e tissues w e r e unavailable for analysis (missing data). 3 Dried tissues w e r e u s e d f o r all tissues o f this clone.

TMS 4488 TMS 30572 TMS]SA 1001 T M S / U 30395 T M S / U 410443 TMS/U 42046

Test clones

Cyanide c o n c e n t r a t i o n s (mg kg -1) in fresh cassava tissues

T A B L E III

43

However, major differences were observed in the HCN content of tissues from sprayed and unsprayed plants. HCN concentrations in the leaves and tuber pulp of sprayed plants were 41--60 and 18--52% more (P < 0.01), respectively, than those of unsprayed stands. R o o t tissues (pulp and peel) from unsprayed stands of TMS 4488 and 30572 contained more cyanide (P < 0.01) than that in similar tissues from sprayed stands of these clones. Tuber periderm from sprayed plots of TMS 4488, 30572 and TMS/U 42046 contained 22--43% more cyanide (P < 0.01) than the pulp. This difference reduced greatly among unsprayed plants of these clones. In one instance (TMS/U 42046), the trend reversed, and the pulp contained 70% more cyanide (P < 0.01) than the peel. DISCUSSION

Over-all mean total cyanide contents of entire plants from sprayed and unsprayed stands were similar. This suggests that the varying pest-infestation levels did not substantially diminish the plant's total synthetic activities for cyanoglucosides. However, individual tissues varied considerably from the whole plant in this respect. For example, leaves of sprayed plants contained 41--60% more total HCN than did those of unsprayed plants of a clone (Table III). Leaves are the main sites of infestation from which CGM depletes chlorophylls > 60% (Ayanru and Sharma, 1983), while CM induce growth abnormalities that cause complete defoliation (Nwanze et al., 1979). It is reasonable, therefore, to expect that in leaves so stressed, there is a diminished synthesis and/or a forced demobilization of cyanogens to other tissues. Although in the case of cassava this is yet to be confirmed, loss of starch and other assimilates from insect-damaged leaves and the pushing out of nitrogenous materials from senescing tobacco leaves stressed by some environmental factors have been reported (Williams, 1955). Our results show that leaves contain the highest concentrations of total cyanide, followed by r o o t and stem tissues, which is in agreement with other studies (Yeoh and Oh, 1979; Pereira et al., 1981). However, total HCN contents in r o o t pulp and peel from unsprayed plants were much higher than those in similar tissues of sprayed plants. Topping of plants and removal of leaves are known to accumulate nitrogenous materials in roots (Watson and Petrie, 1940). Since defoliation and other symptoms on the test plants caused by the pests seem to simulate topping and leaf removal effects, this may partly explain the reported increase of total HCN in root tissues of unsprayed and more heavily defoliated plants. Enhanced total HCN content of tuber pulp relative to that of the peel from unsprayed plants (> 70% for TMS/U 42046) is contrary to the expected pattern of HCN concentration gradients in cassava, whereby HCN content of the peel may be 5--12 times more than that of the pulp (I.I.T.A., 1978). The cause of this alteration in the normal mobilization pattern of cyanogens is unknown. However, since all cassava tissues are capable of

44 synthesizing cyanide (Nartey, 1981), it is concluded that either active synthesis occurred or infestation stimulated mobilization in the pulp. Active synthesis is unlikely as synthesis of cyanide in the r o o t diminishes with age, while demobilization from other tissues into the pulp is possible since basipetal transport of cyanohydrins into cassava roots has been reported (Nartey, 1981). There is no satisfactory explanation as to how the loci of movement of cyanogens and other anabolites are directed in plants. Also, a basis for the control of enzyme systems moderating mobilization activities in plants remains obscure. The mass-flow theory assumes some mobilization force, while active diffusion is suggested to translocate selectively some ions and nitrogenous materials against concentration gradients (Arisz, 1952). Correlative movement of materials from one part of a plant to another may be regulated, in part, by metabolic inhibitors (Nelson et al., 1961), by growth substances such as kinetin (Engelbrecht and Mothes, 1960), and by pathogens (Yarwood and Jacobson, 1955). Secretions of toxic materials by insects into plants are known to induce severe growth abnormalities traceable to changes in growth factors (Leopold, 1964), and to those in the phenol--phenolase system of plants (Miles, 1968). The concept of toxins and inhibitors as moderators of several physiological activities in plants is applicable to a wide range of circumstances. Therefore, it is suggested that CGM- and CM-induced stress in cassava, caused inter alia by the injection of saliva toxins during feeding, may introduce shifting patterns of mobilization whereby cyanogens are translocated preferentially to the pulp rather than to the peel as in healthy plants. The potential of cassava for fuel ethanol production and its other industrial and dietary uses depend on a favourable energy input--output ratio. This ratio m a y be rendered less favourable by added costs due to cyanide detoxication in cassava products, and due to the need to eliminate risks of cyanide poisoning in cassava processing plants. The present study, which shows that enhanced total cyanide content of tuber pulp is caused by pest infestation, provides y e t another compelling reason to rid cassava of CGM and CM. ACKNOWLEDGEMENTS We are indebted to Dr. J.O. O m u e m u of the Bendel State Ministry of Agriculture and Natural Resources (MANR), Benin City, and to Dr. A.R. Opoku, of the Biochemistry Department, University of Benin, Benin City, for the supply of Nuvacron and PNP-glucoside, respectively, used in the study.

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46 Sadik, S., Okereke, O.U. and Hahn, S.K., undated. Screening for Acyanogenesis in Cassava. Int. Inst. Trop. Agric., Ibadan, Nigeria, Tech. Bull. No. 4, 4 pp. Snedecor, G.W. and Cochran, W.G., 1967. Statistical Methods. 6th edn. Iowa State University Press, Ames, 593 pp. Watson, R. and Petrie, A.H.K., 1940. Physiological ontogeny in the tobacco plant. IV. Aust. J. Exp. Biol. Med. Sci., 18: 313--339. Williams, R.T., 1955. Redistribution of mineral elements during development. Annu. R e v . Plant Physiol., 6 : 25--42. Yarwood, C.E. and Jacobson, L., 1955. Accumulation of chemicals in diseased areas of leaves. Phytopathology, 45 : 43--48. Yeoh, H.H. and Oh, H.Y., 1979. Cyanide content of cassava. Malays. Agric. J., 52: 24--28.